The fibroblast growth factor (FGF) family consists of at least nine distinct members (Basilico et al., Adv. Cancer Res. 59:115-165, 1992 and Fernig et al., Prog. Growth Factor Res. 5(4):353-377, 1994) which generally act as mitogens for a broad spectrum of cell types. For example, basic FGF (also known as FGF-2) is mitogenic in vitro for endothelial cells, vascular smooth muscle cells, fibroblasts, and generally for cells of mesoderm or neuroectoderm origin, including cardiac and skeletal myocytes (Gospodarowicz et al., J. Cell. Biol. 70:395-405, 1976; Gospodarowicz et al., J. Cell. Biol. 89:568-578, 1981 and Kardami, J. Mol. Cell. Biochem. 92:124-134, 1990). In vivo, bFGF has been shown to play a role in avian cardiac development (Sugi et al., Dev. Biol. 168:567-574, 1995 and Mima et al., Proc. Nat""l. Acad. Sci. 92:467-471, 1995), and to induce coronary collateral development in dogs (Lazarous et al., Circulation 94:1074-1082, 1996). In addition, non-mitogenic activities have been demonstrated for various members of the FGF family. Non-proliferative activities associated with acidic and/or basic FGF include: increased endothelial release of tissue plasminogen activator, stimulation of extracellular matrix synthesis, chemotaxis for endothelial cells, induced expression of fetal contractile genes in cardiomyocytes (Parker et al., J. Clin. Invest. 85:507-514, 1990), and enhanced pituitary hormonal responsiveness (Baird et al., J. Cellular Physiol. 5:101-106, 1987.)
Several members of the FGF family do not have a signal sequence (aFGF, bFGF and possibly FGF-9) and thus would not be expected to be secreted. In addition, several of the FGF family members have the ability to migrate to the cell nucleus (Friesel et al., FASEB 9:919-925, 1995). All the members of the FGF family bind heparin based on structural similarities. Structural homology crosses species, suggesting a conservation of their structure/function relationship (Ornitz et al., J. Biol. Chem. 271(25):15292-15297, 1996.)
There are four known extracellular FGF receptors (FGFRs), and they are all tyrosine kinases. In general, the FGF family members bind to all of the known FGFRs, however, specific FGFs bind to specific receptors with higher degrees of affinity. Another means for specificity within the FGF family is the spatial and temporal expression of the ligands and their receptors during embryogenesis. Evidence suggests that the FGFs most likely act only in autocrine and/or paracrine manner, due to their heparin binding affinity, which limits their diffusion from the site of release (Flaumenhaft et al., J. Cell. Biol. 111(4):1651-1659, 1990.) Basic FGF lacks a signal sequence, and is therefore restricted to paracrine or autocrine modes of action. It has been postulated that basic FGF is stored intracellularly and released upon tissue damage. Basic FGF has been shown to have two receptor binding regions that are distinct from the heparin binding site (Abraham et al.,. EMBO J. 5(10):2523-2528, 1986.)
It has been shown that FGFR-3 plays a role in bone growth. Mice made homozygous null for the FGFR-3 (xe2x88x92/xe2x88x92) resulted in postnatal skeletal abnormalities (Colvin et al., Nature Genet. 12:309-397, 1996 and Deng et al., Cell 84:911-921, 1996) The mutant phenotype suggests that in normal mice, FGFR-3 plays a role in regulation of chrondrocyte cell division in the growth plate region of the bone (Goldfarb, Cytokine and Growth Factor Rev. 7(4):311-325, 1996). The ligand for the FGFR-3 in the bone growth plate has not been identified.
Although four FGFRs have been identified, all of which have been shown to have functional splice variants, the possibility that novel FGF receptors exist is quite likely. For example, no receptor has been identified for the FGF-8a isoform (MacArthur et al., J. Virol. 69(4):2501-2507, 1995.).
FGF-8 is a member of the FGF family that was originally isolated from mammary carcinoma cells as an androgen-inducible mitogen. It has been mapped to human chromosome 10q25-q26 (White et al., Genomics 30:109-11, 1995.) FGF-8 is involved in embryonic limb development (Vogel et al., Development 122:1737-1750, 1996 and Tanaka et al., Current Biology 5(6):594-597, 1995.) Expression of FGF-8 during embryogenesis in cardiac, urogenital and neural tissue indicates that it may play a role in development of these tissues (Crossley et al., Development 121:439-451, 1995.) There is some evidence that acrocephalosyndactylia, a congenital condition marked by peaked head and webbed fingers and toes, is associated with FGF-8 point mutations (White et al., 1995, ibid.)
FGF-8 has five exons, in contrast to the other known FGFs, which have only three exons. The first three exons of FGF-8 correspond to the first exon of the other FGFs (MacArthur et al., Development 121:3603-3613, 1995.) The human gene for FGF-8 codes for four isoforms which differ in their N-terminal regions: FGF isoforms a, b, e, and f; in contrast to the murine gene which gives rise to eight FGF-8 isoforms (Crossley et al., 1995, ibid.) Human FGF-8a and FGF-8b have 100% homology to the murine proteins, and FGF-8e and FGF-8f proteins are 98% homologous between human and mouse (Gemel et al., Genomics 35:253-257, 1996.)
Heart disease is the major cause of death in the United States, accounting for up to 30% of all deaths. Myocardial infarction (MI) accounts for 750,000 hospital admissions per year in the U.S., with more than 5 million people diagnosed with coronary disease. Risk factors for MI include diabetes mellitus, hypertension, truncal obesity, smoking, high levels of low density lipoprotein in the plasma or genetic predisposition.
Cardiac hyperplasia is an increase in cardiac myocyte proliferation, and has been demonstrated to occur with normal aging in the human and rat (Olivetti et al., J. Am. Coll. Cardiol. 24(1):140-9, 1994 and Anversa et al., Circ. Res. 67:871-885, 1990), and in catecholamine-induced cardiomyopathy in rats (Deisher et al., Am. J. Cardiovasc. Pathol. 5(1):79-88, 1994.) Whether the increase in myocytes originate with some progenitor, or are a result of proliferation of a more terminally differentiated cell type, remains controversial.
However, because infarction and other causes of myocardial necrosis appear to be irreparable, it appears that the normal mechanisms of cardiac hyperplasia cannot compensate for extensive myocyte death and there remains a need for exogenous factors that promote hyperplasia and ultimately result in renewal of the heart""s ability to function.
Bone remodeling is the dynamic process by which tissue mass and skeletal architecture are maintained. The process is a balance between bone resorption and bone formation, with two cell types thought to be the major players. These cells are the osteoblast and osteoclast. Osteoblasts synthesize and deposit matrix to become new bone. The activities of osteoblasts and osteoclasts are regulated by many factors, systemic and local, including growth factors.
While the interaction between local and systemic factors has not been completely elucidated, there does appear to be consensus that growth factors play a key role in the regulation of both normal skeletal remodeling and fracture repair. Some of the growth factors that have been identified in bone include: IGF-I, IGF-II, TGF-xcex21, TGF-xcex22, bFGF, aFGF, PDGF and the family of bone morphogenic proteins (Baylink et al., J. Bone Mineral Res. 8 (Supp. 2):S565-S572, 1993).
When bone resorption exceeds bone formation, a net loss in bone results, and the propensity for fractures is increased. Decreased bone formation is associated with aging and certain pathological states. In the U.S. alone, there are approximately 1.5 million fractures annually that are attributed to osteoporosis. The impact of these fractures on the quality of the patient""s life is immense. Associated costs to the health care system in the U.S. are estimated to be $5-$10 billion annually, excluding long-term care costs.
Other therapeutic applications for growth factors influencing bone remodeling include, for example, the treatment of injuries which require the proliferation of osteoblasts to heal, such as fractures, as well as stimulation of mesenchymal cell proliferation and the synthesis of intramembraneous bone which have been indicated as aspects of fracture repair (Joyce et al. 36th Annual Meeting, Orthopaedic Research Society, February 5-8, 1990. New Orleans, La.).
The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
Within one aspect, the present invention provides An isolated polynucleotide molecule encoding a fibroblast growth factor (FGF) homolog polypeptide selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 82 to nucleotide 621; b) allelic variants of (a); c) polynucleotide molecules that encode a polypeptide that is at least 60% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 28 (Glu) to amino acid residue 207 (Ala); and d) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 6 from nucleotide 82 to nucleotide 621.
In one embodiment, the isolated polynucleotide molecule comprises a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 1 to nucleotide 621 or a nucleotide sequence as shown in SEQ ID NO: 6 from nucleotide 1 to nucleotide 621.
In another embodiment, the isolated polynucleotide molecule comprises a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 82 to nucleotide 621.
In another aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 82 to nucleotide 621; b) allelic variants of (a); c) polynucleotide molecules that encode a polypeptide that is at least 60% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 28 (Glu) to amino acid residue 207 (Ala); and d) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 6 from nucleotide 82 to nucleotide 621; and a transcription terminator.
In another aspect, the present invention provides a cultured cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 82 to nucleotide 621; b) allelic variants of (a); c) polynucleotide molecules that encode a polypeptide that is at least 60% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 28 (Glu) to amino acid residue 207 (Ala); and d) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 6 from nucleotide 82 to nucleotide 621; and a transcription terminator, wherein said cell expresses a polypeptide encoded by the DNA segment.
In another aspect, the present invention provides a method of producing an FGF homolog polypeptide comprising: culturing a cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 82 to nucleotide 621; b) allelic variants of (a); c) polynucleotide molecules that encode a polypeptide that is at least 60% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 28 (Glu) to amino acid residue 207 (Ala); and d) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 6 from nucleotide 82 to nucleotide 621; and a transcription terminator, whereby said cell expresses a FGF homolog polypeptide encoded by the DNA segment; and recovering the FGF homolog polypeptide.
In another aspect, the present invention provides an isolated FGF homolog polypeptide selected from the group consisting of: a) polypeptide molecules comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 28 (Glu) to residue 175 (Met); b) allelic variants of (a); and c) polypeptide molecules that are at least 60% identical to SEQ ID NO: 2 from amino acid residue 28 (Glu) to amino acid residue 175 (Met).
In another aspect, the present invention provides an isolated FGF homolog polypeptide selected from the group consisting of: a) polypeptide molecules comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 28 (Glu) to residue 196 (Lys); b) allelic variants of (a); and c) polypeptide molecules that are at least 60% identical to SEQ ID NO: 2 from amino acid residue 28 (Glu) to amino acid residue 196 (Lys).
In another embodiment, the present invention provides an isolated FGF homolog polypeptide selected from the group consisting of: a) polypeptide molecules comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 28 (Glu) to residue 207 (Ala); b) allelic variants of (a); and c) polypeptide molecules that are at least 60% identical to the amino acids of SEQ ID NO: 2 from amino acid residue 28 (Glu) to amino acid residue 207 (Ala).
In an additional embodiment, the present invention provides an FGF homolog polypeptide further comprising a signal sequence.
In another embodiment, the present invention provides an FGF homolog polypeptide further comprising a signal sequence as shown in SEQ ID NO: 2 from amino acid residue 1 (Met) to amino acid residue 27 (Ala).
The present invention also provides pharmaceutical composition comprising a purified FGF homolog polypeptide, in combination with a pharmaceutically acceptable vehicle.
In another aspect, the present invention provides an antibody that binds to an epitope of a polypeptide molecule comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 1 (Met) to residue 207 (Ala).
In another embodiment, the present invention provides an antibody that binds a polypeptide molecule comprising an amino acid sequence as shown in SEQ ID NO: 2 from residue 28 (Glu) to residue 196 (Lys).
In another aspect, the present invention provides a method of stimulating proliferation of myocytes or myocyte progenitors comprising administering to a mammal in need thereof, an amount of an FGF homolog polypeptide sufficient to produce a clinically significant increase in the number of myocytes or myocyte progenitors in said mammal.
In another embodiment, the present invention provides a method of stimulating proliferation of myocytes or myocyte progenitors, wherein the myocytes or myocyte progenitors are cardiac myocytes or cardiac myocytes progenitors.
In another aspect, the present invention provides a method for ex vivo stimulation of myocyte progenitor cells or myocytes comprising culturing heart tissue cells with an amount of an FGF homolog polypeptide sufficient to produce an increase in the number of myocyte progenitor cells or myocytes in the heart tissue cells cultured in the presence of an FGF homolog polypeptide, as compared to heart tissue myocyte progenitor cells or myocytes cultured in the absence of an FGF homolog polypeptide.
In another embodiment, the present invention provides a method for ex vivo stimulation of myocyte progenitor cells or myocytes, wherein the myocytes or myocyte progenitors are cardiac myocytes or cardiac myocytes progenitors.
In another aspect, the present invention provides a method of delivering an agent or drug selectively to heart tissue comprising: linking a first molecule comprising an FGF homolog polypeptide with a second molecule comprising an agent or drug to form a chimera; and administering the chimera to heart tissue.